Biosynthesis of UDP-GlcNAc(3NAc)A by WbpB, WbpE, and WbpD: Enzymes in the Wbp Pathway Responsible for O-Antigen Assembly in <i>Pseudomonas aeruginosa</i> PAO1

Larkin, Angelyn; Imperiali, Barbara

doi:10.1021/bi900186u

Cited by 45 publications

(73 citation statements)

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“…The high catalytic efficiency of the acetyltransferase drives the production of UDP-diNAcBac by rapidly consuming UDP-4-amino and in turn promotes the conversion of more UDP-GlcNAc to UDP-4-keto. A similar effect can be observed in the biosynthesis of UDP-ManNAc(3NAc)A in P. aeruginosa ; 16 additionally, this type of flux is prevalent in metabolic pathways. For example, glycolytic flux in bacteria utilizes a feed-forward loop where high levels of fructose-1,6-bisphosphate signal for increased activity of glyceraldehyde 3-phosphate dehydrogenase (GAPDH).…”

Section: Discussionsupporting

confidence: 61%

“…Instead, this strain contains a distinct O -linked glycosylation system with a core GalNAc sugar anchoring a branched pentasaccharide. 15 The terminal O -acetylated glucuronic acid sugar (GlcNAc3NAcAOAc) shares homology to a similar pathway in the PAO1 strain of Pseudomonas aeruginosa 16 however these enzymes are absent in the AYE strain of A. baumannii .…”

Section: Introductionmentioning

confidence: 99%

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Biosynthesis of UDP-N,N′-diacetylbacillosamine in Acinetobacter baumannii: Biochemical characterization and correlation to existing pathways

Morrison

Imperiali

2013

Archives of Biochemistry and Biophysics

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The Gram-negative, opportunistic pathogen Acinetobacter baumannii has recently captured headlines due to its ability to circumvent current antibiotic therapies. Herein we show that the multi-drug resistant (MDR) AYE strain of A. baumannii contains a gene locus that encodes three enzymes responsible for the biosynthesis of the highly-modified bacterial nucleotide sugar, UDP-N,N -diacetylbacillosamine (UDP-diNAcBac). Previously, this UDP-sugar has been implicated in the pgl pathway of Campylobacter jejuni. Here we report the overexpression, purification, and biochemical characterization of the A. baumannii enzymes WeeK, WeeJ, and WeeI that are responsible for the production of UDP-diNAcBac. We also demonstrate the function of the phosphoglycosyltransferase (WeeH), which transfers the diNAcBac moiety to undecaprenyl-phosphate. UDP-diNAcBac biosynthesis in A. baumannii is also directly compared to the homologous pathways in the pathogens C. jejuni and Neisseria gonorrhoeae. This work demonstrates for the first time the ability of A. baumannii to generate the highly-modified, UDP-diNAcBac nucleotide sugar found previously in other bacteria adding to the growing list of pathogens that assemble glycoconjugates including bacillosamine. Additionally, characterization of these pathway enzymes highlights the opportunity for investigating the significance of highly-modified sugars in bacterial pathogenesis.

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Section: Discussionsupporting

confidence: 61%

Section: Introductionmentioning

confidence: 99%

Biosynthesis of UDP-N,N′-diacetylbacillosamine in Acinetobacter baumannii: Biochemical characterization and correlation to existing pathways

Morrison

Imperiali

2013

Archives of Biochemistry and Biophysics

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“…There is also a set of three genes in the PSgc7, PSgc9 and PSgc27 gene clusters that have 59%, 82% and 78%, amino acid identity to genes that code for synthesis of UDP-D-GlcNAc3NAc in Pseudomonas aeruginosa [48]. We have no structure for PSgc9 or PSgc27, and the reported Sv7 structure does not seem to relate to the PSgc7 sequence at all as discussed below.…”

Section: Resultsmentioning

confidence: 82%

Diversity in the Major Polysaccharide Antigen of Acinetobacter Baumannii Assessed by DNA Sequencing, and Development of a Molecular Serotyping Scheme

et al. 2013

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We have sequenced the gene clusters for type strains of the Acinetobacter baumannii serotyping scheme developed in the 1990s, and used the sequences to better understand diversity in surface polysaccharides of the genus. We obtained genome sequences for 27 available serovar type strains, and identified 25 polysaccharide gene cluster sequences. There are structures for 12 of these polysaccharides, and in general the genes present are appropriate to the structure where known. This greatly facilitates interpretation. We also find 53 different glycosyltransferase genes, and for 7 strains can provisionally allocate specific genes to all linkages. We identified primers that will distinguish the 25 sequence forms by PCR or microarray, or alternatively the genes can be used to determine serotype by “molecular serology”. We applied the latter to 190 Acinetobacter genome-derived gene-clusters, and found 76 that have one of the 25 gene-cluster forms. We also found novel gene clusters and added 52 new gene-cluster sequence forms with different wzy genes and different gene contents. Altogether, the strains that have one of the original 25 sequence forms include 98 A. baumannii (24 from our strains) and 5 A. nosocomialis (3 from our strains), whereas 32 genomes from 12 species other than A. baumannii or A. nosocomialis, all have new sequence forms. One of the 25 serovar type sequences is found to be in European clone I (EC I), 2 are in EC II but none in EC III. The public genome strains add an additional 52 new sequence forms, and also bring the number found in EC I to 5, in EC II to 9 and in EC III to 2.

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“…A previous report on WlbA from P. aeruginosa suggested that it undergoes an NADH recycling mechanism using α-ketoglutarate as an oxidant to produce 2-hydroxyglutarate (9). Given this, we hypothesized that catalysis by WlbA proceeds through a ping-pong reaction much like that exhibited by glucose-fructose oxidoreductase (20).…”

Section: Resultsmentioning

confidence: 99%

Structural and Functional Studies of WlbA: A Dehydrogenase Involved in the Biosynthesis of 2,3-Diacetamido-2,3-dideoxy-d-mannuronic Acid,

Thoden

Holden

2010

Biochemistry

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2,3-diacetamido-2,3-dideoxy-D-mannuronic acid or ManNAc3NAcA is an unusual dideoxysugar first identified nearly 30 years ago in the lipopolysaccharide of Pseudomonas aeruginosa O:3a,d. It has since been observed in other organisms including Bordetella pertussis, the causative agent of whooping cough. Five enzymes are required for the biosynthesis of UDP-ManNAc3NAcA starting from UDP-N-acetyl-D-glucosamine. Here we describe a structural study of WlbA, the NAD-dependent dehydrogenase that catalyzes the second step in the pathway, namely the oxidation of the C-3′ hydroxyl group on the UDP-linked sugar to a keto moiety and the reduction of NAD+ to NADH. This enzyme has been shown to use α-ketoglutarate as an oxidant to regenerate the reduced dinucleotide. For this investigation three different crystal structures were determined: the enzyme with bound NAD(H), the enzyme complexed with NAD(H) and α-ketoglutarate, and the enzyme complexed with NAD(H) and its substrate (UDP-N-acetyl-D-glucosaminuronic acid). The tetrameric enzyme assumes an unusual quaternary structure with the dinucleotides positioned quite closely to one another. Both α-ketoglutarate and the UDP-linked sugar bind in the WlbA active site with their carbon atoms (C-2 or C3′, respectively) abutting the re face of the cofactor. They are positioned at ~3 Å from the nicotinamide C-4. The UDP-linked sugar substrate adopts a high unusual curved conformation when bound in the WlbA active site cleft. Lys 101 and His 185 most likely play key roles in catalysis.

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Biosynthesis of UDP-GlcNAc(3NAc)A by WbpB, WbpE, and WbpD: Enzymes in the Wbp Pathway Responsible for O-Antigen Assembly in Pseudomonas aeruginosa PAO1

Cited by 45 publications

References 50 publications

Biosynthesis of UDP-N,N′-diacetylbacillosamine in Acinetobacter baumannii: Biochemical characterization and correlation to existing pathways

Biosynthesis of UDP-N,N′-diacetylbacillosamine in Acinetobacter baumannii: Biochemical characterization and correlation to existing pathways

Diversity in the Major Polysaccharide Antigen of Acinetobacter Baumannii Assessed by DNA Sequencing, and Development of a Molecular Serotyping Scheme

Structural and Functional Studies of WlbA: A Dehydrogenase Involved in the Biosynthesis of 2,3-Diacetamido-2,3-dideoxy-d-mannuronic Acid,

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